Volume 56, Issue 4, Pages 1366-1377 (October 1999) Role of MAP kinase pathways in mediating IL-6 production in human primary mesangial and proximal tubular cells  Martin Leonard, Michael P. Ryan, Alan J. Watson, Herbert Schramek, Edel Healy  Kidney International  Volume 56, Issue 4, Pages 1366-1377 (October 1999) DOI: 10.1046/j.1523-1755.1999.00664.x Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 1 Phase contrast microscopy of human proximal tubular (A) and human mesangial cells (B) in culture. Human proximal tubular epithelial cells displayed the typical epithelial cobblestone-like morphology (A). Human mesangial cells appeared as elongated cells in multilayers (B). Magnification (×450). Kidney International 1999 56, 1366-1377DOI: (10.1046/j.1523-1755.1999.00664.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 2 Tumor necrosis factor-α (TNF-α)–induced dose-dependent increase in interleukin-6 (IL-6) production from human proximal tubular (A) and human mesangial (B) cells. Cells were grown to confluence and treated with TNF-α (0.1 to 100 ng/ml) for 24 hours. Cell culture medium was removed and assayed for IL-6 by ELISA, and levels were expressed as fold over basal concentrations (pg/ml). Each value represents the mean ± SEM of 4 to 11 experiments each performed in duplicate. *P < 0.05; **P < 0.01; ***P < 0.001, statistically significant difference compared with control. Kidney International 1999 56, 1366-1377DOI: (10.1046/j.1523-1755.1999.00664.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 3 Inhibition of basal (A and C) and TNF-α (B and D) stimulated IL-6 production from human proximal tubular (A and B) and human mesangial (C and D) cells by the specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. Cells grown to confluence were pretreated with SB203580 (1 to 30 μM) for one hour prior to stimulation with TNF-α (10 ng/ml) for 24 hours. Cell culture medium was removed and assayed for IL-6 by ELISA, and levels in the presence of different inhibitor concentrations were expressed and compared as fold over basal or fold over TNF-α–stimulated concentrations (pg/ml). Each value represents the mean ± SEM of four experiments, each performed in duplicate. *P < 0.05; **P < 0.01; ***P < 0.001; statistically significant difference compared with control. Kidney International 1999 56, 1366-1377DOI: (10.1046/j.1523-1755.1999.00664.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 4 TNF-α–induced stimulation of p38 MAPK phosphorylation (activation) in human proximal tubular (A) and human mesangial (B) cells, which were not affected by SB203580. Confluent cells, serum deprived for 24 hours, were pretreated with SB203580 (10 μM) for one hour followed by TNF-α (10 ng/ml) stimulation for 15 minutes. Whole cell extracts subjected to SDS-PAGE were probed using Western blotting and antibodies directed against the phosphorylated or whole cell p38 MAPK. The bands were visualized by ECL. Phosphorylated p38 MAPK is shown in the upper panels, and whole cell p38 MAPK is shown in the lower panels. Representative blots from one of three separate experiments are shown. Kidney International 1999 56, 1366-1377DOI: (10.1046/j.1523-1755.1999.00664.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 5 Inhibition by SB203580 of the TNF-α–stimulated increase in p38 MAPK activity in human proximal tubular epithelial cells. Confluent cells, serum deprived for 24 hours were pretreated with SB203580 (10 μM) for one hour followed by TNF-α (10 ng/ml) stimulation for 15 minutes. p38 MAPK was immunoprecipitated from whole cell extracts, and activity was measured as the incorporation of a radiolabeled phosphate into an artificial substrate ATF2. The incorporation was visualized by autoradiography after SDS-PAGE. Kidney International 1999 56, 1366-1377DOI: (10.1046/j.1523-1755.1999.00664.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 6 Inhibition of basal (A and C) and TNF-α (B and D)–stimulated IL-6 production from human proximal tubular (A and B) and mesangial (C and D) cells by the specific MEK1 inhibitor PD98059. Cells grown to confluence were pretreated with PD98059 (0.01 to 10 μM) for one hour prior to stimulation with TNF-α (10 ng/ml) for 24 hours. Cell culture medium was removed and assayed for IL-6 by ELISA, and levels in the presence of different inhibitor concentrations were expressed and compared as fold over basal or fold over TNF-α–stimulated concentrations (pg/ml). Each value represents the mean ± SEM of four to eight experiments each performed in duplicate. *P < 0.05; **P < 0.01; ***P < 0.001; statistically significant difference compared to control. Kidney International 1999 56, 1366-1377DOI: (10.1046/j.1523-1755.1999.00664.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 7 TNF-α–induced stimulation of ERK1,2 phosphorylation (activation) in human proximal tubular (A) and mesangial (B) cells, which were inhibited by the specific MEK1 inhibitor PD98059. Confluent cells, serum deprived for 24 hours, were pretreated with PD98059 (10 μM) for 1 hour followed by TNF-α (10 ng/ml) stimulation for 15 minutes. Whole cell extracts subjected to SDS-PAGE were probed using Western blotting and antibodies directed against the phosphorylated form of ERK1,2 MAPK or whole cell ERK2. The bands were visualized by ECL. Phosphorylated ERK1,2 MAPK is shown in the upper panels, and whole cell ERK2 is shown in the lower panels. Representative blots from one of three separate experiments are shown. Kidney International 1999 56, 1366-1377DOI: (10.1046/j.1523-1755.1999.00664.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 8 Further inhibition of basal (A and C) and TNF-α (B and D)–stimulated IL-6 production from human proximal tubular (A and B) and human mesangial (C and D) cells by a combination of the MAPK inhibitors SB203580 and PD98059. Cells grown to confluence were pretreated with PD98059 (10 μM) and SB203580 (10 μM) for one hour prior to stimulation with TNF-α (10 ng/ml) for 24 hours. Cell culture medium was removed and assayed for IL-6 by ELISA, and levels in the presence of inhibitors were expressed and compared as fold over basal or fold over TNF-α–stimulated concentrations (pg/ml). Each value represents the mean ± SEM of four to eight experiments each performed in duplicate. *P < 0.05; **P < 0.01; statistically significant difference compared to each inhibitor added alone. Kidney International 1999 56, 1366-1377DOI: (10.1046/j.1523-1755.1999.00664.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 9 Proposed hypothesis for the regulation of TNF-α–induced IL-6 production in renal cells. Kidney International 1999 56, 1366-1377DOI: (10.1046/j.1523-1755.1999.00664.x) Copyright © 1999 International Society of Nephrology Terms and Conditions